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Ziegler, J.; Abel S. Analysis of amino acids by HPLC/electrospray negative ion tandem mass spectrometry using 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) derivatization Amino Acids 46, 2799-2808, (2014) DOI: 10.1007/s00726-014-1837-5

A new method for the determination of amino acids is presented. It combines established methods for the derivatization of primary and secondary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) with the subsequent amino acid specific detection of the derivatives by LC–ESI–MS/MS using multiple reaction monitoring (MRM). The derivatization proceeds within 5 min, and the resulting amino acid derivatives can be rapidly purified from matrix by solid-phase extraction (SPE) on HR-X resin and separated by reversed-phase HPLC. The Fmoc derivatives yield several amino acid specific fragment ions which opened the possibility to select amino acid specific MRM transitions. The method was applied to all 20 proteinogenic amino acids, and the quantification was performedusing l-norvaline as standard. A limit of detection as low as 1 fmol/μl with a linear range of up to 125 pmol/μl could be obtained. Intraday and interday precisions were lower than10 % relative standard deviations for most of the amino acids. Quantification usingl-norvaline as internal standard gave very similar results compared to the quantificationusing deuterated amino acid as internal standards. Using this protocol, it was possible to record the amino acid profiles of only a single root from Arabidopsis thaliana seedlings and to compare it with the amino acid profiles of 20 dissected root meristems (200 μm).

Fellenberg, C.; Ziegler, J.; Handrick, V.; Vogt, T. Polyamine homeostasis in wild type and phenolamide deficient <i>Arabidopsis thaliana</i> stamens. Front Plant Sci. doi: 10.3389/fpls.2012.00180 3, 180, (2012)

Polyamines (PAs) like putrescine, spermidine, and spermine are ubiquitous polycationic molecules that occur in all living cells and have a role in a wide variety of biological processes. High amounts of spermidine conjugated to hydroxycinnamic acids are detected in the tryphine of Arabidopsis thaliana pollen grains. Tapetum localized spermidine hydroxycinnamic acid transferase (SHT) is essential for the biosynthesis of these anther specific tris-conjugated spermidine derivatives. Sht knockout lines show a strong reduction of hydroxycinnamic acid amides (HCAAs). The effect of HCAA-deficient anthers on the level of free PAs was measured by a new sensitive and reproducible method using 9-fluorenylmethyl chloroformate (FMOC) and fluorescence detection by HPLC. PA concentrations can be accurately determined even when very limited amounts of plant material, as in the case of A. thaliana stamens, are available. Analysis of free PAs in wild type stamens compared to sht deficient mutants and transcript levels of key PA biosynthetic genes revealed a highly controlled regulation of PA homeostasis in A. thaliana anthers.
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